Genetic Methods 1

Question 1

What are restriction endonucleases

Where do they orginate from

Answer 1

Originate in bacteria, which use restriction enzymes to protect themselves from foreign DNA

Cleave the covalent bonds of the sugar phosphate backbone at recognition sites

They are used in vitro for cloning, mapping, and ligation

Question 2

What are the blunt ends of an endonuclease

Answer 2

DNA ligase will join the fragments together
They cut in the same position, there is no sequence specificity, just need 2 backbones, so they are less accurate

Question 3

What is are palindromic sequence

Answer 3

There are two types: sticky 5 prime ends or sticky 3 prime ends. This is where they cut at different places so that there is an overhang on whichever end and hydrogen bonds are possible hybridization is possible with complementary sequence and DNA ligase forms covalent bonds along backbones

Question 4

What do shorter recognition sequences tend to have

Answer 4

More frequent cut sites

Question 5

How do you caculate the average fragment length

Answer 5

So if it it is four bases than you would do 1/4 X1/4X1/4X1/4 = 1/256 meaning you will see this every 1/256 bases

Question 6

What is a complete digest

Answer 6

cleavage has occured at every DNA site recognizable by the enzyme

Question 7

What is partial digest

Answer 7

Not all available restriction sites have been cut (Possibility of complete digest of a small population)

Question 8

What is important in considering the digest

Answer 8

The timing of exposure to restriction endonuclease helps determine the amount of digestion

Question 9

What is gel electrophoresis seperate fragments based on

Answer 9

Basis of size/length

Question 10

What is the difference between agarose

Answer 10

Agarose separates larger molecules
Polyacrylamide can separate molecules 10-500 nucleotides in length

Question 11

What is DNA hybridization

Answer 11

Process of combining two single-stranded DNA molecules to form one double-stranded DNA molecule through base pairing

Question 12

What is denaturation vs renaturation

Answer 12

Denaturation is breaking down base interactions in dsDNA to form ssDNA
Renaturation: Reverting ssDNA back to dsDNA

Question 13

What is Tm

Answer 13

Melting temperature at which of the dsDNA has neatured into ssDNA

Question 14

What are factors that affect Tm

Answer 14

GC content C-G form 3 hydrogen bonds while A-T form 2

Salt concentration: DNA is negatively charged. Positive ions in higher salt concentration Na+ “Shield” the negative charges, making the duplex more stable

pH-high pH, alkaline, negative OH-
OH ion concentration is increased when pH is high and it removes hydrogen from nitrogenous bases, breaking down the integrity

Question 15

Can denatured DNA strands renature

Answer 15

Yes if they can form a complementary double helix. However, some mismatches are tolerated depending on the conditions of hybridization

Question 16

What is low stringency

Answer 16

Make it easier for DNA to bind even if they don’t match perfectly. These conditions are high salt and low temperature

Question 17

What is high stringency

Answer 17

It makes it harder for DNA to bind if there are mismatches, and it must have a perfect match on renaturation, low salt, high temperature

Question 18

What are some factors of DNA probes

Answer 18

Single stranded
Complementary to the sequence of interest
Length of probe can vary (100 to 1000 nucleotides)
Labelled with a marker for visualization (radioactive isotope)
Will hybridize to the sequence of interest if it is present in the sample

Question 19

What is the Southern blot

Answer 19

Used to detect the presence or absence of DNA sequence in DNA samples using DNA to look for DNA

Question 20

What are the steps in the southern blot

Answer 20

1. Digest DNA with restriction endonuclease
2. Gel electrophoresis
3. Denature DNA with a strong alkaline solution
4. Transfer DNA from gel to membrane (blotting)
5. Incubate the membrane with the radioactively labelled DNA probe (low stringency), you want the strands to bind even if there are some mismatches
6. Wash of unhybridized probes at high stringency, so if they are not bound, they will get washed
7. Place the membrane on an X-ray film for autoradiography (visualization)

Question 21

What is Northern blot

Answer 21

Used to study gene expression by detecting the level of mRNA in a sample

Question 22

What are the steps of Northern blot

Answer 22

1. Isolate mRNA from tissues
2. Gel electrophoresis
3. Transfer RNA from gel to membrane
4. Incubate the membrane with radioactively labelled cDNA probe
5. Wash off unhybridized probes high stringency
6. Visualize probes hybridized to DNA using autoradiography

Question 23

What is the Western plot used for

Answer 23

Used to detect the presence of an individual protein in a protein mixture

Question 24

What are the steps of the Western blot

Answer 24

1. Sample preparation
2. Gel electrophoresis
3. Blotting - transfer to membrane
4. Antibody probing (primary and secondary probe)
5. Detection and imaging

Question 25

What is the difference between the SDS page and the Native page

Answer 25

SDS page denatures proteins Separation in the gel solely by the size of the protein Native page does not denature proteins Separation in the gel depends on Charge Size 3D structure

Question 26

What is PCR

Answer 26

Replicates ("amplifies") target DNA in vitro Can get millions of copies from little starting material in a short amount of time

Question 27

What are the three main steps of PCR and what temperature they use

Answer 27

Denature 94 degrees 5 mins for the first round 20 seconds subsequent rounds Anneal 50-60 2 mins Elongate 72 degrees 2-5 mins

Question 28

What are some characterstics of primers for PCR

Answer 28

Complementary to sequence flanking the region to be amplified (target sequence) 2 primers per reacation: forward and reverse roughly 16 to 26 bp in length GC content is between 40-60% (3' ends needs to be C or G) Tm between 65 and 75 within 5 degress of each other Not complementary to one another

Question 29

What are componets of PCR reaction

Answer 29

DNA to be amplified dNTPs Taq (Therums aquaticus) Polymerase Primers Buffer + MgCl2

Question 30

What should the temperature be for PCR

Answer 30

Set annealing temperature a few degrees below (3-5) primer melting temperature too low and primer mismatch occurs Set higher denaturation temperature for DNA with high GC content

Question 31

What is the optimization in regards to time

Answer 31

Set extension step for 1-2 minutes per kilobase (Kb) of product Increase the time of denaturation for GC-rich target DNA

Question 32

What is optimization of PCR for oligonucleotide primers

Answer 32

Designe primers to be complementary to the 3' ends of the sense and anti-sense strands of template DNA Design primers to have similar melting temperatures Avoid primer-dimers and secondary structure formation

Question 33

What is the optimization of the PCR buffer solution

Answer 33

MgCl2 in the buffer creates a stable environment for the polymerase to function. Mg2+ is a Taq cofactor Higher Mg2+ increases the error rate of DNA polymerase

Question 34

What are you going to see with the different bands on a electrophoresis

Answer 34

WT G:C is uncut and then heterozygous you would see mutiple bands of different lengths Homozygous you would see shorter cuts